Molecules 2011, 16, 403-411; doi:10.

3390/molecules16010403
OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Herbicidal Activity of Peumus boldus and Drimys winterii

Essential Oils from Chile
Mercedes Verdeguer 1, David Garca-Relln 1, Herminio Boira 1, Eduardo Prez 2,
Sandra Gandolfo 2 and Mara Amparo Blzquez 3,*
1

Instituto Agroforestal Mediterrneo, Universidad Politcnica de Valencia, Camino de Vera s/n, C.P.
46022 Valencia, Spain; E-Mails: merversa@doctor.upv.es (M.V.);
davidgr82@hotmail.com (D.G.-R.); hboira@eaf.upv.es (H.B.)
Centro Regional de Estudios en Alimentos Saludables, CREAS, Blanco 1623 Of.1402, Valparaso,
Chile; E-Mails: eduardoperez@creas.cl (E.P.); gandolfosandra@gmx.de (S.G.)
Departamento de Farmacologia, Facultat de Farmcia, Universitat de Valncia, Avda Vicent Andrs
Estells s/n, C.P. 46100 Burjassot, Spain

* Author to whom correspondence should be addressed; E-Mail: amparo.blazquez@uv.es;

Tel.: + 34 96 354 49 45; Fax: + 34 96 354 49 43.
Received: 29 November 2010; in revised form: 17 December 2010 / Accepted: 7 January 2011 /
Published: 10 January 2011

Abstract: The essential oil composition of Peumus boldus and Drimys winterii was
analyzed by means of capillary GC-FID and GC-MS. More than 96% of the total oil
components (43 and 54 compounds, respectively) were identified, with ascaridole
(51.17 9.51), p-cymene (16.31 2.52) and 1,8-cineole (14.45 2.99) as the main
compounds in P. boldus and -eudesmol (21.65 0.41), followed of elemol (12.03 0.34)
and terpinen-4-ol (11.56 1.06) in D. winterii. The herbicidal activity was tested against
Amaranthus hybridus and Portulaca oleracea. P. boldus essential oil was the most
phytotoxic against both weeds, inhibiting seed germination and seedling growth at all
concentrations assayed (0.1251 L/mL). D. winterii essential oil did not show any effect
on A. hybridus germination and only affected P. oleracea germination at the highest
concentration. The results suggest the possible use of the essential oil from P. boldus as a
natural herbicide.
Keywords: essential oils; phytotoxicity; germination; seedling growth; monoterpenes

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1. Introduction
Weeds are a small group of different plants from the taxonomic point of view. They display a great
adaptability to different habitats, taking advantage of the favorable conditions that occur in agricultural
systems and competing with crops. Indiscriminate application of synthetic herbicides has contributed
to increased resistance in weeds, also leading to gradual degradation of soil and the environment, and
to hazards to human health. The secondary metabolites of plant species offer an excellent potential to
develop new herbicide formulations, or as a guide towards identifying active components to obtain
natural herbicides [1,2], thus numerous studies have been done with plant-derived compounds, in order
to obtain synthetic herbicide substitutes for weed control [3].
Peumus boldus Mol. (Monimiaceae), is a native tree from the central region of Chile, being part of
the sclerophyllous forests, characteristic of the Mediterranean climate. It is the source of the known
crude drug Boldo folium, commonly used as a medicinal plant in Chile and reported as a herbal
remedy in several pharmacopeias [4]. Infusion of boldo leaves is recommended for the treatment of
gastrointestinal spasms, dyspectic and hepatobiliary disorders [5]. The main bioactive compounds of
boldo leaves are flavonoids, alkaloids and essential oils. Several studies have demonstrated the
antioxidant activity of flavonoids and alkaloids, particularly the aporphine alkaloid boldine [6-8]. The
antimicrobial, fungicidal or antihelmintic effects of ascaridole, the main compound in the essential oil,
is also reported [9,10], but to our knowledge, no assays of the herbicidal activity of the essential oil
have been carried out so far.
Drimys winterii J.R.Forst. & G.Forst (Winteraceae) is a native tree of Chile that belongs to the
Magallanian and Valdivian temperate rain forests. The indigenous Mapuches consider it a sacred tree
with antiinflammatory, antitumoural, antibacterial and insecticidal properties. Its leaves contain
flavonoids, tannins, terpenoids and also essential oil [11]. Thus, the essential oils of both native species
could be promising sources of products to evaluate new activities such as the phytotoxic effect on seed
germination and seedling growth of weeds.
Amaranthus hybridus and Portulaca oleracea are two annual weeds of tropical and subtropical
crops with an extensive world distribution. They have become cosmopolitan weeds distributed in a
wild range of soils and climates. In Mediterranean crops these weeds show summer phenology. The
aims of the present study were to determine the composition of the essential oils from two endemic
plants from Chile and to compare their phytotoxic activity against A. hybridus L. and. P. oleracea L
with that of plants from the Mediterranean area that we had previously tested [12].
2. Results and Discussion
The phytotoxic effects of P. boldus and D. winterii essential oils obtained from Chilean plants have
been investigated against A. hybridus and P. oleraceae, two major weeds in summer crops in the
Mediterranean area,. Results obtained can be explained by the very different qualitative and
quantitative chemical composition of both essential oils. The essential oil composition of P. boldus and
D. winterii analyzed by GC-FID and GC-MS is shown in Table 1, where the identified compounds are
classified by phytochemical groups and listed in order of their elution on a methylsilicone
HP-1 column.

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Table 1. Constituents of the essential oils from Peumus boldus, and Drimys winterii by GC
and GC-MS analysis.
Compound
Monoterpene hydrocarbons
-thujene
-pinene
camphene
sabinene
-pinene
myrcene
-phelladrene
-3-carene
-terpinene
p-cymene
limonene
-phellandrene
cis-ocimene
-terpinene
terpinolene
p-cymenene
1.3.8-p-menthatriene
Oxygenated monoterpenes
dehydro-1,8-cineole
1,8-cineole
cis-sabinene hydrate
fenchone
trans-sabinene hydrate
linalool
dehydro-sabina ketone
cis-p-menth-2-en-1-ol
trans-pinocarveol
trans-p-menth-2-en-1-ol
camphor
sabina ketone
pinocarvone
-terpineol
terpinen-4-ol
thuj-3-en-10-al
cryptone
-terpineol
myrtenal
cis-piperitol
myrtenol
trans- piperitol
ascaridole
cis-piperitone epoxide
trans-piperitone epoxide
bornyl acetate
thymol

RI
930
939
952
976
979
991
1005
1011
1019
1027
1032
1033
1042
1062
1089
1090
1112
992
1034
1070
1088
1097
1100
1121
1122
1140
1141
1146
1160
1169
1169
1179
1185
1186
1191
1195
1196
1197
1207
1242
1259
1262
1288
1293

Peumus boldus
(mean s.e.)
19.71 2.94
0.27 0.04
0.11 0.01
1.20 0.25
0.18 0.02
t
t
0.30 0.03
16.31 2.52
0.31 0.13
0.42 0.00
t
0.64 0.29
74.77 4.26
0.09 0.02
14.45 2.99
0.56 0.28
t
0.51 0.19
t
0.83 0.23
1.50 0.26
0.13 0.02
t
0.30 0.11
0.64 0.11
2.15 0.33
t
t
0.06 0.01
0.09 0.03
1.11 1.33
51.17 9.51
0.76 0.20
t
0.08 0.01
0.06 0.01

Drimys winterii
(mean s.e.)
16.74 2.43
0.05 0.02
0.93 0.23
2.97 0.68
2.68 0.58
0.99 0.15
0.25 0.09
1.70 0.19
0.05 0.02
2.46 0.48
0.47 0.05
3.30 0.20
0.91 0.04
13.95 1.23
0.13 0.01
t
t
0.07 0.01
t
0.44 0.06
11.56 1.06
1.61 0.11
t
0.14 0.01
-

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Table 1. Cont.

p-cymen-7-ol
carvacrol
Sesquiterpene hydrocarbons
-elemene
-cedrene
-caryophyllene
trans--farnesene
-acoradiene
-curcumene
-curcumene
bicyclogermacrene
Oxygenated sesquiterpenes
4-epi-cis-dihydroagarofuran
italicene ether
elemol
E-nerolidol
spathulenol
rosifoliol
5-epi-7-epi--eudesmol
-oplopenone
epi-cedrol
10-epi--eudesmol
-eudesmol
-eudesmol
-eudesmol
7-epi--eudesmol
-bisabolol
-bisabolol
drimenol
drimenin
Aromatic compounds (C6-C3)
safrole
eugenol
methyl eugenol
myristicin
elemicin
Others
3-octanol
1.4-dione-2-cyclohexene
2-nonanone
3-octanol acetate
2-undecanone
TOTAL IDENTIFIED

1295
1302
1392
1411
1419
1459
1465
1481
1484
1496
1499
1536
1555
1566
1578
1602
1607
1609
1614
1621
1639
1656
1659
1662
1674
1687
1763
1951
1287
1360
1406
1522
1560
993
1015
1093
1128
1295

0.12 0.02
0.24 0.15
0.27 0.05
0.12 0.03
0.15 0.02
1.20 0.18
1.20 0.18
0.34 0.03
0.06 0.01
0.07 0.00
0.24 0.03
96.90 1.06

2.89 0.18
0.14 0.02
0.30 0.04
0.19 0.00
0.15 0.04
t
1.91 0.14
0.17 0.03
0.04 0.01
57.82 1.38
0.34 0.02
0.12 0.01
12.03 0.34
0.36 0.01
t
0.08 0.00
t
0.75 0.01
1.86 0.06
21.65 0.41
7.27 0.35
7.35 0.14
0.12 0.04
4.61 0.22
0.07 0.02
1.18 0.04
0.06 0.01
4.83 0.42
0.09 0.01
1.09 0.10
t
3.66 0.32
t
t
t
t
96.24 0.28

RI: retention index relative to C8-C32 n-alkanes on the HP-1 column. Peak area percentages were
calculated in GC analysis on an apolar HP-1 column. t: trace amounts <0.03. Values are means
standard error of three samples.

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P. boldus essential oil is rich in monoterpene compounds, which account for more than 95% of the
total oil composition. The monoterpene hydrocarbons fraction (19.71 2.94) is constituted mainly by
p-cymene (16.31 2.52). Of the twelve monoterpene hydrocarbons identified, only this compound and
sabinene (1.20 0.25) reached percentages higher than 1%. Twenty five compounds were identified in
the oxygenated monoterpenes fraction (74.77 4.26). The monoterpene endoperoxide ascaridole
(51.17 9.51) followed by 1,8-cineole (14.45 2.99), terpinen-4-ol (2.15 0.33) and transpinocarveol (1.50 0.26) were the main compounds. Only two oxygenated sesquiterpenes, spathulenol
(0.12 0.03) and -oplopenone (0.15 0.02) have been identified in P. boldus essential oil. This
essential oil is also characterized by the absence of sesquiterpene hydrocarbons. On the other hand,
only methyl eugenol, that reached a percentage around 1%, was found among the aromatic
compounds. The high herbicidal activity showed by P. boldus essential oil correlates with the fact that
a high percentage of oxygenated monoterpenes is linked to a potent phytotoxic activity [13-15].
Nevertheless, it is interesting to note that according to previous results [12], not only the
monoterpene compounds may be responsible for germination inhibition [16,17], because previous
assays with Eucalyptus camaldulensis essential oil, rich in the oxygenated sesquiterpene spathulenol
(41.46 3.94), showed that it also completely inhibited A. hybridus and P. oleracea seed germination
and seedling growth [12].
Fifty-four compounds were identified in D. winterii essential oil, accounting for 96% of the total oil
composition. The monoterpene hydrocarbons with 12 identified compounds almost reached 17%
(16.74 2.42), among them p-cymene only reached 0.08 in one sample, being detected in the other
two as trace amounts (<0.03%). The higher percentages corresponded to -terpinene (3.30 0.20),
sabinene (2.97 0.68), -pinene (2.68 0.58), limonene (2.46 0.48) and -terpinene (1.70 0.19).
Substantial differences on oxygenated monoterpenes were observed between the two analyzed species.
This fraction (13.95 1.23), represented the second most important group on D. winterii essential oil,
with terpinen-4-ol (11.56 1.06) followed of -terpineol (1.61 0.11) as the main compounds. With
the exception of 1,8-cineole, cis and trans-sabinene hydrate, linalool, terpinen-4-ol and -terpineol,
which were identified in both essential oils, the oxygenated monoterpenes found in P. boldus were not
detected in D. winterii essential oil.
The greatest differences comparing both species show up in the sesquiterpene fraction. The eight
sesquiterpene hydrocarbons found in D. winterii were absent in the three samples of P. boldus. With
17 identified compounds, oxygenated sesquiterpenes constituted the predominant group in D. winterii
essential oil. This fraction (57.82 1.38) contained mainly elemol (12.03 0.34), -bisabolol
(4.61 0.22) and eudesmol compounds: -eudesmol (21.65 0.41), -eudesmol (7.35 0.14) and
-eudesmol (7.27 0.35).
Finally, relative large amounts of myristicin (3.66 0.32) and eugenol (1.09 0.10), aromatic
compounds (C6-C3) (4.83 0.42) were found in D. winterii. From this fraction only methyl eugenol
was detected in P. boldus essential oil. The essential oil of D.winterii, which also contained high
percentages of oxygenated compounds was not active against A. hybridus germination and seedling
growth. No significant differences were found between control and all concentrations applied.
Nevertheless this essential oil was effective reducing P. oleracea germination at the higher
concentrations assayed (Table 2). On the other hand D. winterii essential oil showed phytotoxic effects
against P. oleraceae seedlings growth all concentrations assayed (Figure 1).

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Table 2. Effect of Peumus boldus and Drimys winterii essential oils on Portulaca oleracea
seeds germination.
Treatment
CONTROL
0.125 L/mL
0.250 L/mL
0.5 L/mL
1 L/mL

Peumus boldus
essential oil
71.0 2.9 a
0.0 0.0 b
0.0 0.0 b
0.0 0.0 b
0.0 0.0 b

Drimys winterii
essential oil
71.0 2.9 ab
74.0 5.8 a
53.0 6.0 b
30.0 6.1 c
27.5 9.9 c

Values are means standard error of five replicates of 20 seeds each after 14 days of incubation.
Within each species, different letters indicate that means are not equal at the 95% level of
probability (Fishers least significant difference test, LSD).

Figure 1. Seedling length (mm) (mean SE) measured for 14 days of P. oleracea control
or treated with D. winterii essential oil at 0.125, 0.25, 0.5 and 1 L/mL concentrations.

3. Experimental
3.1. Plant material
Leaves of wild Peumus boldus Molina and Drimys winterii J.R.Forst. & G.Forst. were collected in
the winter of 2010 (May) at Comuna of Hualqui (365736S; 725548W, VIII Region, Chile) and
in Quilpu (3303S; 7127W, V Region, Chile) respectively. Both areas are characterized by a
temperate Mediterranean climate.
Mature seeds of the annual weeds Amaranthus hybridus L. and Portulaca oleracea L. were
collected from parent plants growing in citrus orchards of the province of Valencia (Spain), in October
2005 and August 2008, respectively. The plants were dried during 15 days at room temperature,

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afterwards the seeds were extracted. Uniform healthy seeds were selected and stored at 4 C until
germination tests.
3.2. Oil isolation
The fresh material was subjected to hydro-distillation for three hours in a Clevenger-type apparatus,
yielding (v/w) % after three distillations 1.25 0.20 for P. boldus essential oils and 0.22 0.02 for D.
winterii essential oils. All samples were stored at 4 C until analysis, upon which they were either
diluted to 1% (v/v) in dichloromethane or their herbicidal potential was tested.
3.3. GC and GC-MS analyses
Gas chromatography was performed using a Perkin-Elmer Clarus 500GC apparatus equipped with a
flame ionization detector (FID), a Hewlett-Packard HP-1 (cross-linked methyl silicone) capillary
column (30 m long and 0.2 mm i.d., with 0.33 m film thickness). The column temperature program
was 60 C during 5 min, with 3 C/min increases to 180 C, then 20 C/min increases to 280 C, which
was maintained for 10 min. The carrier gas was helium at a flow-rate of 1 mL/min. Both the FID
detector and injector port temperature were maintained at 250 and 220 C, respectively. Gas
chromatography-mass spectrometry analysis were carried out with a Varian Saturn 2000 equipped with
a Varian C.S VA-5MS capillary column (30 m long and 0.25 mm i.d. with 0.25 m film thickness).
The same working conditions used for GC and split mode injection (ratio 1:25) were employed. Mass
spectra were taken over the m/z 28400 range with an ionizing voltage of 70 eV. Kovats retention
index were calculated using co-chromatographed standard hydrocarbons. The individual compounds
were identified by MS [18] and their identity was confirmed by comparison of their RIs, relative to C8C32 n-alkanes, and by comparing their mass spectra and retention times with those of authentic samples
or with data already available in the NIST 98 library and in the literature.
3.4. Biological assay
Seed germination and growth seedling tests. Sets of 20 seeds each with five replicates per treatment
were put in Petri dishes (9 cm diameter) between two layers of filter paper (Whatman No.1) wetted
with 4 mL of distilled water for germination. Essential oils of P. boldus or D. winterii were added at
volumes of 0 (control), 0.5, 1, 2, and 4 L. Based on previous assays [12], A. hybridus and P. oleracea
seeds were incubated alternating 30.0 0.1 C 16 hr in light and 20.0 0.1 C 8 hr in dark. To
evaluate the allelopathic potential of the essential oils, germination and seedling length data were
recorded after 3, 5, 7, 10 and 14 days.
3.5. Statistical analyses
Tests were conducted in a randomised complete design with five replications. Data were submitted
to analysis of variance (ANOVA). Percentage values were arcsin transformed. The means were
compared using Fishers least significant difference (LSD) test (P 0.05).

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4. Conclusions
The present study has examined the phytotoxic effects of the essential oils of P. boldus and D.
winterii two medicinal tree from Chile, against A. hybridus and P. oleracea, two serious weeds in
Mediterranean summer crops. The essential oil of P. boldus was the most effective, completely
inhibiting both A. hybridus and P. oleraceae seed germination and seedling length at all the
concentrations applied (0.1251 L/mL). D. winteri showed selective activity, depending on the weed
assayed. Its essential oil was not active against A. hybridus germination and only significantly reduced
P. oleracea germination (57.7 and 61.3%) at the highest concentrations (0.5 and 1 L/mL). Both
species provided essential oils rich in oxygenated compounds, P. boldus oxygenated monoterpenes
(66.3977.61%) and D. winterii oxygenated sesquiterpenes (55.6560.37%). The results corroborated
previous studies suggesting that a high percentage of oxygenated monoterpenes is correlated with
potent phytotoxic activity. Our in vitro studies suggest a possible and new alternative use of P. boldus
essential oil in herbicidal formulations, although further experiments involving field conditions are
necessary to confirm the herbicidal potential of P. boldus.
Acknowledgements
Research performed under an International Cooperation Project (number A/024753/09), financed by
Agencia Espaola de Cooperacin Internacional para el Desarrollo (AECID).
References
1. Duke, S.O.; Romagni, J.G.; Dayan, F.E. Natural products as sources for new mechanisms of
herbicidal action. Crop Prot. 2000, 19, 583-589.
2. Vyvyan, J.R. Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron 2002,
58, 1631-1636.
3. Tehmina, A.; Stevenson, P.; Hall, D.; Bajwa, R. Allelophatic potential of Helianthus annuus L.
(sunflower) as natural herbicide. In Establishing the Scientific Base, Proceedings of the Fourth
World Congress on Allelopathy; Charles Sturt University: Wagga Wagga, NSW, Australia, 2126
August 2005.
4. Speisky, H.; Cassel, B.K. Boldo and boldine: An emerging case of natural drug development.
Pharmacol. Res. 1994, 29, 1-10.
5. Simirgiotis, M.J.; Schmeda-Hirschmann, G. Direct identification of phenolic constituents in Boldo
Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode
array detection and electrospray ionization tandem mass spectrometry. J. Cromatogr. A 2010,
1217, 443-449.
6. Silva, E.; Jopia, M.; Edwards, A.M.; Lemp, E.; de la Fuente, J.R.; Lissi, E. Protective effect of
boldo and tea infusions on the visible light-mediated pro-oxidant effects of vitamin B2, riboflavin.
Photochem. Photobiol. 2002, 75, 585-590.
7. Milin, L.; Estells, R.; Abarca, B.; Ballesteros, R.; Sanz, M.J.; Blzquez, M.A. Reactive oxygen
species (ROS) generation inhibited by aporphine and phenanthrene alkaloids semi-synthesized
from natural boldine. Chem. Pharm. Bull. 2004, 52, 696-699.

Molecules 2011, 16

411

8. Quezada, N.; Asencio, M.; Del Valle, J.M.; Aguilera, J.M.; Gmez, B. Antioxidant activity of
crude extract, alkaloid fraction, and flavonoid fraction from Boldo (Peumus boldus Molina) leaves.
J. Food Sci. 2004, 69, 371-376.
9. Vila, R.; Valenzuela, L.; Bello, H.; Caigueral, S.; Montes, M.; Adzet, T. Composition and
antimicrobial activity of the essential oil of Peumus boldus leaves. Planta Med. 1999, 65, 178-179.
10. Bittner, M.; Aguilera, M.A.; Hernndez, V.; Arbert, C.; Becerra, J.; Casanueva, E. Fungistatic
activity of essential oils extracted from Peumus boldus Mol., Laureliopsis philippiana (Looser)
Schodde and Laurelia sempervirens (Ruix y Pav.) Tul (Chilean Monimiaceae). Chilean J. Agric.
Res. 2009, 69, 30-37.
11. Muoz-Concha, D.; Vogel, H.; Razmilic, I. Variacin de compuestos qumicos en hojas de
poblaciones de Drimys ssp (Magnoliophyta: Winteraceae) en Chile. Rev. Chil. Hist. Nat. 2004, 77,
43-50.
12. Verdeguer, M.; Blzquez, M.A.; Boira, H. Phytotoxic effects of Lantana camara, Eucalyptus
camaldulensis and Eriocephalus africanus essential oils in weeds of Mediterranean summer crops.
Biochem. Syst. Ecol. 2009, 37, 362-369.
13. Vokou, D.; Douvli, P.; Blionis, G.J.; Halley, J.M. Effects of monoterpenoids, acting alone or in
pairs, on seed germination and subsequent seedling growth. J. Chem. Ecol. 2003, 29, 2281-2301.
14. Kotan R.; Cakir, A.; Dadasoglu, F.; Aydin, T.; Cakmakci, R.; Ozer, H.; Kordali, S.; Mete,
E.;Dikbas, N. Antibacterial activities of essential oils and extracts of Turkish Achillea, Satureja
and Thymus species against plant pathogenic bacteria. J. Sci. Food Agric. 2010, 90, 145-160.
15. Rolim de Almeida, L.F.; Frei, F.; Mancini, E.; De Martino, L.; De Feo, V. Phytotoxic activities of
Mediterranean essential oils. Molecules 2010, 15, 4309-4323.
16. Dudai, N.; Ben-Ami, M.; Chaimovich, R.; Chaimovitsh, D. Essential oils as allelopathic agents:
bioconversion of monoterpenes by germinating wheat seeds. Acta Hort. 2004, 629, 505-508.
17. Armirante, F.; De Falco, E.; De Feo, V., De Martino, L.; Mancini, E., Quarante, E. Allelopathic
activity of essential oils from Mediterranean Labiatae. Acta Hort. 2006, 723, 347-352.
18. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass
Spectrometry; Allured Publishing Corporation: Carol Stream, IL, USA, 2007.
Sample Availability: Samples of the compounds are available from the authors.
2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).